Abstract

Reversible injury is a dynamic condition in tissues exposed to multiple harm situations including ischemia and post-ischemic recovery, in which the skeletal striated muscle tissue evidences histopathological characteristics to adapt to damage. During the inflammation and the regeneration phases of healing, the myocytes show morphological changes with an apparently complete recovery at the end of the treatment. Though the satellite cells play a key role in the recovery, not in all cases the regeneration is completely achieved. That is why in this research we measured the histopathological patterns evidenced by enzyme histochemistry and morphometric measurements, during the spontaneous recovery of skeletal muscle fibers underwent to short periods of ischemia of one and three hours and prolonged periods of reperfusion up to 32 days (768 hours). The selected muscles were the extensor carpi radialis longus and the soleus from Wistar rats. There were significant differences in the distribution of type of fibers, shape, size, leukocyte infiltration, necrosis and presence of central nuclei. The soleus muscle adapts better during early reperfusion than extensor carpi radialis longus does. Nevertheless, both muscles evidenced an incomplete recovery at day 32. The extrapolation of these findings suggests the importance of further following up of patients and the improvement of recovery the muscle function after surgery and/or other harm conditions.

Keywords

Ischemia; Reperfusion; Morphometry; Myocytes; Skeletal
muscle

Introduction

Orthopedic surgeries such as arthroscopic procedures imply the use
of a pneumatic or a manual tourniquet. Also, controlling bleeding due
to accidents occurring in distant places and treatments for joint
diseases or contractures requires a tourniquet. The use of the
tourniquet entails reversible injury triggered by ischemic conditions
during determined periods of time [1-3]. Arthroscopic procedures, for
instance, are performed during 15 to 60 minutes, while to transport an
injured person from far places to the nearest health institution, can
require two to three hours.

In those cases, the muscles under the level of compression suffer the
associated damage due to the ischemic condition followed by
reperfusion injury [4,5]. The lesion patterns studied in myocytes after
induced ischemia of two to four hours comprise edema, atrophy and
changes in their surrounding connective tissue [5-8]. The recovery of
the histological and functional characteristics of the muscles depends
on their extracellular matrix and the type of fibers. The molecular
mechanisms of adaptation lead to muscle fibers to change their
oxidative or glycolytic predominance in the muscle [4,9,10].

More than two decades ago, it was considered that irreversible
injury mainly occurs during ischemic conditions longer than five
hours [11,12]. However, since the last 20 years, the evidence has
pointed out that ischemic conditions less than three hours caused by
an acute compartmental syndrome, tourniquet or artery clamp, for example, lead to necrosis [3,13]. The stem cells of the skeletal striated
muscle tissue, called satellite cells, are postnatal myogenic precursors
that can regenerate the myocytes [14]. Then, the recovery of the muscle
tissue underwent to reversible injury should lead to a proper
rearrangement spontaneously. Some studies regarding a complete
recovery of skeletal muscle include the injection of stromal
mesenchymal cells into the injured muscle, in which the myocytes
achieve a complete recovery 14 days after reperfusion [15]. This is an
important aspect to consider for treatment after injury. A different
research based on mouse models evidenced an apparently total
functional recovery by day 56 after the ischemic condition. This was
performed with a manual tourniquet without control of the pressure of
ischemia [16].

In the present study, through conventional and enzymatic
histochemistry, were analyzed the changes of lesion and recovery
caused by an induced ischemic injury in skeletal muscle fibers.
Treatments or additional interventions were not applied to describe the
histopathological characteristics of the spontaneous recovery and/or
adaptation in the skeletal striated muscle tissue throughout postischemic
lesion until day 32.

Materials and Methods

Experimental animals

All procedures performed during this research involving lab
animals, were in accordance with the ethical standards of the Animal
Ethics Committee of Universidad del Valle and International Guidelines for Care and Use of Animals [17]. The Ethics Committee
approved this research through the act 036-15. This study was
conducted by using 42 male adult rats, Wistar albino strain, genus
Rattus, 12 months old and weighing 450 to 500 grams.

The rats were obtained from the bioterium of the Universidad del
Valle, which provided to the animals with food and water ad libitum.
These rats were handled considering the Institutional Standard Guides
to the Use of Laboratory Animals. The extensor carpi radialis longus
and soleus were suitable muscles for this research, due to their
glycolytic and oxidative predominance of their muscle fibers
respectively [4,10,18]. The surgical access is relatively easy, mainly for
the forelimb. This let us to reduce the surgical time required for the
dissection of both muscles, important for a proper histochemical
staining.

Surgical procedure

The rats were divided into one control group and 20 experimental
groups of ischemia of one hour and three hours followed by
reperfusion of 0 hours, 1 hour, 16 hours, 24 hours, 2 days (48 hours), 4
days (96 hours), 8 days (192 hours), 16 days (384 hours), 24 days (576
hours) and 32 days (768 hours), respectively. These periods of
reperfusion were selected based on previous studies focused on morphological and physiological recovery of the skeletal muscle tissue
after ischemic injury [4,5,19,20]. The total sample size of 42 rats was
determined based on 21 groups, significant level of 0.05 and confident
level of 0.95.

Under anesthetic condition with Isofluorane® Baxter and
pentobarbital (Penthal® - INVET 64.8 mg/ml) 4-5 mg/100 grams per
weight, two pneumatic tourniquets were set to each rat. One
tourniquet was set in the proximal region of the right forelimb near to
the shoulder, and the other one in the proximal region of the left
hindlimb near to the hip. The complete description of both tourniquets
can be founded in our previous work [8]. The pressure of ischemia was
adjusted at 260 mmHg during one or three hours regarding to each
experimental group. This pressure is suitable to perform a complete
ischemic condition; due to microcirculatory blood flow is not present
at 230 mmHg ± 20 mmHg [1,4,15,21]. Once the time of ischemia finished, both tourniquets were removed. Postsurgical analgesia with
tramadol 2 mg/100 mg was administered to each rat.

Histochemical stains

Once the time of reperfusion was over, an overdose of pentobarbital
was injected intraperitoneally to perform euthanasia. During the
following 5 to 10 minutes we verify the death signs (respiratory failure,
absence of heart beat and opaque cornea) and subsequently we
dissected soleus and extensor carpi radialis longus muscles. The
samples were directly fixed by freezing using 2-methyl butane
(isopentane) cooled in liquid nitrogen, stored at -70°C and cut on
cryostat Leica Jung Frigocut 2800N at -20°C and 16 ums. The stain
techniques were enzyme histochemistry for NADH-TR (nicotinamide
adenine dinucleotide tetrazolium reductase), mATPase pH 4.6 (myosin
adenosine triphosphatase), and conventional histochemistry for H&E
(hematoxylin and eosin).

For NADH-TR, the tissues were incubated during 60 minutes at
37°C in Tris 0.2M solution (Sigma-Aldrich®) pH 7.4 with HCl 0.1 M,
Nitro-Blue Tetrazolium 10 mg (Sigma-Aldrich®) and Beta
Nicotinamide Adenine dinucleotide 8 mg (Sigma-Aldrich®) in distilled
water pH 7.2. Oxidative fibers stained dark and glycolytic stained clearer. For mATPase stain, the samples were pre-incubated in ATP I
during 25 minutes at 22°C. Then, ATP II solution by using adenosine 5
´triphosphate (Sigma-Aldrich®) 15 mg was prepared, and adjusted to
pH 10.6. The slides were incubated during 40 minutes at 37°C and
fibers were revealed with ammonium sulfide 1% for 15 seconds. The
acidic pre-incubation lead to a stronger reaction in the slow twitchfibers
while fast twitch fibers stain pale [22,23].

Morphometric analysis

The oxidative- slow twitch fibers and glycolytic- fast twitch fibers
were measured by image processing and segmentation of regions, to
determine the areas of interest through image analysis techniques
[24,25]. The other parameters measured through semiautomatic
counting were:

• Leukocyte infiltration in muscle fibers

• Round shape

• Increasing in the size of myocytes

• Decreasing in the size of myocytes

• Presence of central nuclei

• Necrosis, evidenced by a pale stain, fragmentation of cytoplasm
and loss of cellular morphology.

These patterns and measurements of the areas were performed by
using Image Pro Plus v7.0 (Media Cybernetics Inc.). The
measurements of each parameter were carried out to 174 images per
muscle, randomly selected from a total amount of 404 microscopic
fields in 40X magnification to determine cell characteristics. This size
of images per muscle was calculated based on a confidence interval of
1.96. Statistical analysis consisted of comparisons between one and
three hours of ischemia during reperfusion through Mann Whitney
test. The periods of ischemia/reperfusion that showed significant
differences were analyzed by multiple comparisons test, as nonparametric
Bonferroni post-ANOVA. Kruskal – Wallis test was used
for comparisons among the reperfusion groups, this is the nonparametric
equivalent of the one-way analysis of variance. SPSS
version 22 (IBM Analytics Corp.) was the statistical software used for
this analysis.

This study described morphological characteristics of limb skeletal
myocytes, during the recovery after short and prolonged periods of
ischemic injury. The stain for NADH-TR lead to the identification of
oxidative and glycolytic muscle fibers, while mATPase pH 4.6 permits
the recognition of slow twitch (type I) and fast twitch fibers (Type IIB).
Both stain techniques complement each other in the analysis of the histopathological patterns observed in a muscle biopsy.

Results

The control group evidenced a normal predominance in the
glycolytic fibers and fast-twitch fibers in the extensor carpi radialis
longus muscle. In contrast, the soleus muscle has oxidative fibers
predominance and a slow-twitch fibers majority (Table 1). For both
muscles, the myocytes in the cross-sectional microscopic fields
evidenced hexagonal-like shape and multiple nuclei adjacent to the
sarcolemma. The above mentioned abnormal parameters in the
morphometric analysis were negative for both control groups (Figures
1A-1C).

Distribution area of fiber type (ums)

Parameters/muscles

Extensor carpi radialis longus (x͂)

Soleus (x͂)

Control

Reperfusion after Ischemia 1h

Reperfusion after Ischemia 3h

Control

Reperfusion after Ischemia 1h

Reperfusion after Ischemia 3h

Oxidative (NADH-TR)

9263

14237.3

13009.4

36212

27046.7

11912.3

Glycolytic (NADH-TR)

30552

11038.7

11312.4

0

5327.27

5327.84

Slow twitch (mATPase pH 4, 6)

1051

3178.48

4001.7

9076

21237.92

21686.37

Fast twitch (mATPase pH 4, 6)

8125

15140,04

76,92,486

0

1591,19

393,69

Table 1: Note the total tendency of the areas of each type of fibers in both muscles in comparison to the control groups.

Figure 1: Parameters measured in muscle fibers during reperfusión.
A-B-C Control group. Normal characteristics in the extensor carpi
radialis longus observed on each staining. Glycolytic (NADH) and
fast twitch (mATPase pH 4.6) fibers predominance. D-E-F Short
ischemia and prolonged reperfusion. NADH staining evidences
stronger reactive fibers in comparison to the control group. The fast
twitch predominance observed in mATPase staining is conserved,
and changes in the shape and size (circle) are evident in the HE. GH-
I Prolonged ischemia and short reperfusion. Smaller fibers are
observed and there is an increasing in their surrounding connective
tissue. Leukocytes are also present (circle) J-K-L Control group.
Normal characteristics in the soleus muscle observed on each
staining. Oxidative (NADH) and slow fast twitch (mATPase pH4.6)
fibers predominance. M-N-O Short ischemia and prolonged
reperfusion. A decreased in the reaction of the fibers to NADH is
observed. One fast twitch fiber (yellow circle) is present in the
microscopic field for mATPase. Smaller fibers (black circle) are
observed in HE in comparison to the control group. P-Q-R
Prolonged ischemia and short reperfusion. There is a
disorganization of the cytoplasm observed in the three staining.
More than one fast twitch fiber (yellow circle) is present in the
microscopic field for mATPase. Magnification 40x. Scale Bar: 25 μ.

Extensor carpi radialis longus

The experimental groups evidenced mainly during prolonged
reperfusion, an increasing in the oxidative fibers areas in comparison
to the control group. This characteristic suggests a change in the
predominance of muscle fibers during reperfusion after being exposed
to ischemic conditions of one hour and three hours p- value 0.045
(Figures 1D, 1G and 2A). The areas of slow twitch fibers were also
superior in comparison to the control group, mainly during
reperfusion after three hours of ischemia (Table 1). This tendency in
the mechanism of contraction seen through mATPase pH 4.6 suggests
a functional adaptation during the recovery p-value 0.027 (Figures 1E and 3A).

Figure 2: Measurements of the areas of oxidative and glycolytic
fibers during reperfusión. X-axis: Time; Y-axis: Area (ums). A. The
extensor carpi radialis longus muscle evidenced during early
reperfusion changes in the normal glycolytic tendency, which is
similar throughout reperfusion until 32 days. B. In the soleus
muscle the areas of oxidative fibers are near to the control group.
There was an increasing in the glycolytic fibers that modifies the
oxidative tendency of this muscle.

In contrast, the areas of glycolytic fibers decreased during
reperfusion after both periods of ischemia, which corresponds with the
oxidative tendency pattern seen through NADH-TR p-value 0.034
(Figures 1D, 1G and 2A). To respect to the fast twitch fibers, this
mechanism of contraction tended to increase during reperfusion after
one hour of ischemia, which suggests that the muscle function can be
conserved and recovered after short ischemic injuries (Table 1). During
reperfusion after three hours of ischemia the total area of this type of
fibers was minor but comparable the total area of the control group
(Figures 1E and 3A).

Figure 3: Measurements of the areas of slow twitch fibers and fast
twitch fibers during reperfusión. X-axis: Time; Y-axis: Area (ums).
A. The extensor carpi radialis longus muscle evidenced a general
increasing of the slow twitch fibers, while fast twitch fibers were not
severely affected during reperfusion after the ischemic conditions of
one hour and three hours. B. The soleus muscle conserved a
predominance of slow twitch fibers throughout reperfusion.
Nonetheless, the presence of fast twitch fibers was observed during
reperfusion after one and three hours of ischemia.

Regarding to the other parameters measured through
semiautomatic counting, the presence of leukocyte infiltration was a
common characteristic in the surrounding connective tissue of the
cells. Though it was present during reperfusion after both periods of
ischemia, in this analysis we prefer to measure the presence of immune
cells in the muscle fibers. This is important because these cells
influence the recovery and/or scar tissue formation processes
consistent with the severity of the injury. Leukocytes were mainly
present during reperfusion after three hours of ischemia but during
early reperfusion after one hour of ischemia p-value 0.025. On the
other hand, fibers with round shape were mainly observed during early
periods of reperfusion after both periods of ischemia p-value 0.025.
The increasing in the size of muscle fibers was evidenced during
prolonged reperfusion after three hours of ischemia p-value 0.025. The
decrease in the size of fibers was present during early reperfusion
especially after the ischemic condition of three hours p-value 0.000.
Central nuclei were significant during prolonged reperfusion after one
hour and three hours of ischemia, p-value 0.000, while few necrotic
fibers were present during early reperfusion after both periods of
ischemia (Figures 1F, 1I and 4).

Figure 4: Measurements of abnormal parameters observed in
muscle fibers during reperfusión. X-axis: Time; Y-axis: Number of
fibers. A. The shape and size of the myocytes are the parameters
mainly altered during prolonged reperfusion after one hour of
ischemia. B. Throughout reperfusion after three hours of ischemia
the measured parameters are present. The increase in fibers with
round shape and fibers with central nuclei are notable during early
and prolonged reperfusion.

Soleus

The morphometric analysis evidenced a decrease of the oxidative
predominance during reperfusion, mainly throughout reperfusion
after three hours of ischemia, p-value 0.026. During reperfusion after
one hour of ischemia, the amount of oxidative fibers was major or near
to the control group areas (Figures 1M, 1P and 2B). The slow twitch
fibers evidenced increases along reperfusion after one and three hours
of ischemia p-value 0.032 (Figures 1N, 1Q and 3B). These changes
suggest that the soleus muscle adapts well to reperfusion injury to
maintain its function and mechanism of contraction (Table 1).

The areas of glycolytic fibers were mainly present during prolonged
reperfusion after the ischemic conditions of one and three hours, pvalue
0.027 (Figures 1M and 1P). These areas did not exceed the areas
measured for oxidative fibers. Likewise, a low quantity of areas of fast
twitch fibers was observed throughout reperfusion after one and three
hours of ischemia (Table 1). This characteristic suggests adaptation mechanisms of the soleus muscle to tolerate the ischemic injuries
(Figures 1N, 1Q and 2B).

Regarding the other histopathological patterns, the presence of
leukocyte infiltration was mainly observed during reperfusion after
three hours of ischemia p-value 0.001. Round shape fibers were
observed throughout reperfusion after one hour of ischemia and were
mainly present during prolonged reperfusion after three hours of
ischemia p-value 0.000. In contrast, the increasing in the size of fibers
was observed during prolonged reperfusion after one hour of ischemia
p-value 0.022, while few muscle fibers with decreasing on their size
were observed in the microscopic fields. Central nuclei were not a
significant change during reperfusion after one hour, but after three
hours of ischemia these cells were still present during prolonged
reperfusion p-value 0.001. Finally, few necrotic fibers were present
during reperfusion after one and three hours of ischemia (Figures 1O,
1R and 5).

Figure 5: Measurements of abnormal parameters observed in
muscle fibers during reperfusión. X-axis: Time; Y-axis: Number of
fibers. (A) The increasing in the size of fibers and round shape are
the notable changes along reperfusion after one hour of ischemia.
(B) During reperfusion after three hours of ischemia round shape
and leukocyte infiltration in myocytes are the patterns still present
in the prolonged periods.

Discussion

The recovery of striated muscle tissue undergone reversible injury,
such as short ischemic conditions, entails inflammation, changes in
metabolic substrates and decreased in ATP levels, among other factors
[19,26]. Upon releasing the stimulus that caused ischemia, the blood
flow is present and injury due to reperfusion occurs [27]. Short periods
of reperfusion can cause apparently reversible lesions. Carmo-Araujo et al. described the effects of induced ischemia of four hours in soleus,
followed by one hour of reperfusion. The main changes resemble the
findings of the present study that include altered shape of the
myocytes, decreasing in size and increasing in the space occupied by
the connective tissue [5].

Our previous studies evidenced changes in the areas occupied by
the muscle fibers and the intramuscular extracellular matrix [7,8].
Both patterns have a different tendency in both muscles. At day 32 of
reperfusion, the area of the connective tissue measured in the extensor
carpi radialis longus was superior to the area of the myocytes. In
contrast, at the same day, the area of the connective tissue measured in
the soleus muscle was inferior in comparison to the control group [8].
These findings suggest that the increasing in the muscle fibers leads the
muscle to maintain its contractile function. These results are consistent
to the studies that describe a better recovery in the soleus muscle than
in glycolytic predominance muscles [4].

Skeletal muscles contain a high proportion of a determined type of
fiber depending on their function and location. For instance, the
predominance of extensor muscles is of type IIb fibers, whereas flexor
muscles are of oxidative fibers [28,29]. Each kind of muscle fibers
respond in a different way when underwent to ischemia and
reperfusion injury. Chan et al. observed injury due to three hours of
reperfusion in type 2 fibers that endured two hours of ischemia [30].
Likewise, studies conducted by Walters et al. demonstrated that skeletal
muscles with oxidative predominance tolerate prolonged periods of
ischemia and reperfusion. Their results determined that one of the
contributing factors in the damage and the recovery of the muscle
fibers is their predominance in the muscle [4]. This characteristic is
associated with the strong ability of the soleus muscle to adapt to
reperfusion injury [4].

The research published by Sassoli and co-workers considered cell
differentiation and function of connective tissue cells in the expression
of matrix metalloproteinases [31]. Ghaly and Marsh described an
increasing in the concentration of leukocytes, myeloperoxidase, and
chemotactic factors, during early hours of reperfusion [32]. A gradual
decreasing is present after three days of reperfusion, with a rising in
monocyte infiltration and oxidative activity of macrophages during the
following periods [32]. The type of fibers and the intramuscular
connective tissue influence the recovery of skeletal muscle during
reperfusion [33,34]. In this research, although there were
inflammatory changes and presence of leukocytes during reperfusion,
there was no evidence of fibrosis that led to a replacement of muscle
fibers by connective tissue.

The parameters of regeneration as central nuclei are one of the
interesting findings still present in prolonged reperfusion up to day 32.
Itoh et al. evidenced central nuclei in the tibialis anterior that was
underwent to one hour of ischemia with a gradual decreasing until day
14 of reperfusion [35]. Saclier et al. used the term regenerated
myofiber, to refer to the myocytes with central nuclei [36]. The
presence of central nuclei indicates a stage of differentiation and
recovery in the striated skeletal muscle tissue underwent to injury
[35,36]. In the present research, the soleus and the extensor carpi
radialis longus evidenced different patterns of regeneration for each
muscle. Likewise, both muscles evidenced distinct changes in size and
shape of muscle fibers, possibly related with oedema and protein
degradation after short periods of ischemia, as have seen in muscle
fibers obtained from postsurgical patients [37].

The follow up after clinical procedures that require the use of the
tourniquet are mainly focused on the possible complications and the
treated region, such as the joints [38]. Based on the present study, the
structures under the level of compression, for example the muscles, are
suitable to be also included in this follow up and upcoming therapies.
The muscles are also susceptible to the ischemic and reperfusion
conditions, so they evidence changes during the post-surgical recovery
that can influence the final functional outcomes of patients, being this
aspect the main reason for a more complete follow up.

This research provided a histopathological evaluation of skeletal
striated muscle tissue during prolonged reperfusion. The evaluated
patterns have influence in the postsurgical recovery and explain the
impairment in muscle function that occurs in several clinical cases.
Based on the findings of the present research up to day 32 of
reperfusion, abnormal patterns are still present in muscle fibers mainly
in the extensor carpi radialis longus, which are aspects to consider in
the postoperative and/or post-traumatic follow up of patients.

Conclusion

Injury after short ischemic conditions up to three hours can alter the
normal characteristics of the skeletal striated muscle tissue in the rat
model established in this study. During early and prolonged
reperfusion, the affected muscles evidence patterns of adaptation to
endure damage due to ischemia, but mainly due to reperfusion. One of
them is the predominance of the muscle fibers that change throughout
reperfusion to maintain the contractile function of the muscle.

Conflict of Interest

The authors declared no potential conflicts of interest with respect
to the research or publication of this article.

Acknowledgements

The authors want to acknowledge to the Laboratory of Histology of
Universidad del Valle, Teblami Research Group, Nhora Holguín,
Martha Ceballos and Henry Vidal of Universidad del Valle, for their
valuable contributions to this study.